Biofabrication of advanced functional materials using bacterial cellulose scaffolds

ABSTRACT

A biofabrication method for producing cellulose-based materials. The method includes providing active cellulose-producing bacteria such as  Gluconacetobacter  and culturing media in a container; combining organic additives or inorganic additives with the active cellulose-producing bacteria in the container to produce a cellulose hydrogel matrix composed of entangled bacteria-produced cellulose nanofibers; controlling a concentration of the additives in the cellulose hydrogel matrix; and exposing the cellulose hydrogel matrix in selected thermal environment to create a biofabricated functional material.

GOVERNMENT INTEREST

The embodiments herein may be manufactured, used, and/or licensed by orfor the United States Government without the payment of royaltiesthereon.

BACKGROUND Technical Field

The embodiments herein generally relate to biofabrication techniques,and more particularly to biofabrication techniques to create compositematerials.

Description of the Related Art

Plant-derived cellulose materials have been used as metal organicframeworks for energy storage applications, and, when carbonized,metal-free catalysts and filtration systems. Traditional fabricationapproaches typically require significant processing steps to render thematerial useful for a device. To achieve nanoparticle-incorporatedbacterial cellulose, prior strategies have included addition ofnanoparticles to media during static culture or post-growth particlesynthesis through alkaline precipitation (e.g., for iron oxides) orchemical oxidation of bacterial cellulose for reactivity with silversalts to form silver nanoparticle coatings. Conventionally, carbonnetworks (i.e., graphene-like materials) are obtained from bacterialcellulose after it is cultivated and purified. To dope the carbon withother elements, conventional methodologies use absorption ofpolysaccharide-staining dyes (e.g., methylene blue, Congo red, both ofwhich are sulfur and nitrogen-containing compounds) prior to pyrolyzingthe material.

It is generally challenging to fabricate 3-dimensional porous scaffoldsfor high-surface area materials. Conventional techniques generallyrequire chemical processing of plant biomass to obtain cellulosenanofibers followed by their chemical crosslinking. Moreover,functionalization of cellulose materials typically requires many stepsand the use of harsh chemicals. Limited conventional methods exist fortethering functional groups to polymer/cellulose scaffolds. Furthermore,these techniques are generally difficult to control with nano/microprecision. Additionally, conventional chemical processing techniquestypically use harsh chemicals and conditions as well as strictenvironments (e.g., cleanroom). Moreover, it is generally difficult touniformly impregnate porous materials with nanoparticles orfunctionalization reagents in a top-down way. Accordingly, there is aneed for a new biofabrication technique to produce composite materialsthat overcomes the limitations of the conventional solutions.

SUMMARY

In view of the foregoing, an embodiment herein provides a method ofproducing cellulose-based structures, the method comprising providingbacteria in a container; providing media in the container to sustain thebacteria; combining additives with the bacteria to produce a cellulosenanofiber; controlling a concentration of the additives in the cellulosenanofiber to create a uniform distribution of the additives in thecellulose nanofiber; and processing the cellulose nanofiber at aselected temperature to create a composite material. The bacteria maycomprise Gluconacetobacter. The additives may comprise organicadditives.

The organic additives may comprise any of compounds, click handleanalogs, carbon/oxide particles, biopolymers, and catalytic enzymes. Thebiopolymers may comprise any of proteins, carbohydrates, and nucleicacids. The additives may comprise inorganic additives. The inorganicadditives may comprise any of metal nanoparticles, metal oxides, andmetal salts. The cellulose nanofiber may be modified during productionthrough any of diffusion, binding, and surface modification of fibersconstituting the cellulose nanofiber. The controlling of theconcentration of the additives may occur by purifying the cellulosenanofiber by solvent exchange to diffuse away extra additives. Thecontrolling of the concentration of the additives may occur byevaporating the cellulose nanofiber to enhance the concentration of theadditives within the cellulose nanofiber. The processing of thecellulose nanofiber at the selected temperature may comprisefreeze-drying the cellulose nanofiber. The freeze-drying of thecellulose nanofiber may create a dehydrated material. The processing ofthe cellulose nanofiber at the selected temperature may compriseperforming pyrolysis on the cellulose nanofiber. The performing of thepyrolysis on the cellulose nanofiber may create a carbonized material.

Another embodiment provides a biofabrication method comprising providingGluconacetobacter in a container; providing culturing media in thecontainer; coincubating additives with the Gluconacetobacter in thecontainer to produce a cellulose hydrogel; controlling a concentrationof the additives in the cellulose hydrogel to form a composite precursormaterial; and thermally processing the composite precursor material. Theadditives may comprise a chemical compound or salt as a source ofnitrogen, iron, phosphorous, sulfur, or other chemical element ofinterest for impregnation or nanoparticle formation. The method maycomprise forming a metallized aerogel from the thermally processedcomposite precursor material. The method may comprise forming a dopedelectrocatalyst from the thermally processed composite precursormaterial. The coincubating of the additives with the Gluconacetobacterin the container may occur at a temperature range between approximately20-30° C.

Another embodiment provides a biofabrication method for producingcellulose-based materials, the method comprising providing activecellulose-producing bacteria and culturing media in a container;combining organic additives or inorganic additives with the activecellulose-producing bacteria in the container to produce a cellulosehydrogel matrix composed of entangled bacteria-produced cellulosenanofibers; controlling a concentration of the additives in thecellulose hydrogel matrix; and exposing the cellulose hydrogel matrix inselected thermal environment to create a biofabricated functionalmaterial.

These and other aspects of the embodiments herein will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following descriptions, while indicatingexemplary embodiments and numerous specific details thereof, are givenby way of illustration and not of limitation. Many changes andmodifications may be made within the scope of the embodiments hereinwithout departing from the spirit thereof, and the embodiments hereininclude all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments herein will be better understood from the followingdetailed description with reference to the drawings, in which:

FIG. 1 is a schematic diagram illustrating an apparatus for producingcellulose-based structures, according to an embodiment herein;

FIG. 2 is a schematic diagram illustrating a workflow for producingcellulose-based structures, according to an embodiment herein;

FIG. 3A is a flow diagram illustrating a biofabrication method,according to an embodiment herein;

FIG. 3B is a flow diagram illustrating a method of forming a metallizedaerogel, according to an embodiment herein;

FIG. 3C is a flow diagram illustrating a method of forming a dopedelectrocatalyst, according to an embodiment herein;

FIG. 4 is a flow diagram illustrating a biofabrication method forproducing cellulose-based materials, according to an embodiment herein;

FIG. 5 are microscopic images of cellulose hydrogels bonded tonanoparticles, according to an embodiment herein;

FIG. 6A is a scanning electron micrograph of strontium titanate (ST)powder, according to an embodiment herein;

FIG. 6B is a is a scanning electron micrograph of a sinteredST-cellulose composite material, according to an embodiment herein;

FIG. 6C is a graph illustrating stoichiometric ratios of the atomicpercentage of strontium (Sr) to titanium (Ti), according to anembodiment herein;

FIG. 6D is an image of a bulk dielectric material, according to anembodiment herein;

FIG. 7A is a graph illustrating timecourse monitoring ofGluconacetobacter productivity, according to an embodiment herein;

FIG. 7B is a graph illustrating measurements of a cellulose biomass,according to an embodiment herein;

FIG. 7C is a graph illustrating cellulose mass vs. residual glucose,according to an embodiment herein;

FIG. 8A is a graph illustrating measurements of cellulose mass,according to an embodiment herein;

FIG. 8B are images of cellulose hydrogel materials, according to anembodiment herein;

FIG. 8C is a scanning electron micrograph of a cellulose hydrogelmaterial, according to an embodiment herein;

FIG. 8D are additional images of carbonized cellulose materials,according to an embodiment herein;

FIG. 8E is a scanning electron micrograph of a carbonized material,according to an embodiment herein;

FIG. 9A is a graph illustrating the composition of cellulose derivedmesoporous carbons, according to an embodiment herein;

FIG. 9B is a scanning electron micrograph of Fe—S,P,N carbons, accordingto an embodiment herein;

FIG. 9C is a graph illustrating X-ray photoelectron spectroscopy (XPS)data for urea-doped and undoped cellulose derived carbons, according toan embodiment herein; and

FIG. 10 are cyclic voltammagrams obtained on cellulose-derived carbonswhere various chemicals are provided during growth to impart particularelements to the resulting material, according to an embodiment herein.

DETAILED DESCRIPTION

The embodiments herein and the various features and advantageous detailsthereof are explained more fully with reference to the non-limitingembodiments that are illustrated in the accompanying drawings anddetailed in the following description. Descriptions of well-knowncomponents and processing techniques are omitted so as to notunnecessarily obscure the embodiments herein. The examples used hereinare intended merely to facilitate an understanding of ways in which theembodiments herein may be practiced and to further enable those of skillin the art to practice the embodiments herein. Accordingly, the examplesshould not be construed as limiting the scope of the embodiments herein.

It will be understood that when an element or layer is referred to asbeing “on”, “connected to”, or “coupled to” another element or layer, itmay be directly on, directly connected to, or directly coupled to theother element or layer, or intervening elements or layers may bepresent. In contrast, when an element or layer is referred to as being“directly on”, “directly connected to”, or “directly coupled to” anotherelement or layer, there are no intervening elements or layers present.It will be understood that for the purposes of this disclosure, “atleast one of X, Y, and Z” may be construed as X only, Y only, Z only, orany combination of two or more items X, Y, and Z (e.g., XYZ, XYY, XZ,ZY, YZ, XX, YY, ZZ, etc.).

The embodiments herein provide a biofabrication workflow to assembleprecursors of functional materials using an in situ-produced bacterialcellulose scaffold and added components. Biocompatible fabricationmethodologies include the innate cellulose production characteristics ofbacterial species Gluconacetobacter and principles ofbiologically-mediated precision binding and hierarchical assembly. Sincethe biofabrication steps occur autonomously and in an ambientenvironment (cellulose accumulation, binding interactions forself-assembly, passive diffusion and incorporation in aqueous solutionand room temperature), the techniques provided by the embodiments hereinsimplify the fabrication process for several materials (e.g., withdielectrics and electrocatalysts, for example). Conventional workflowsmay require user-based fabrication of a polymeric aerogel framework,surface chemistry and chemical grafting protocols, and organic/aqueoussolvent exchanges with challenges in scalability, shaping, and compositeuniformity. Additionally, in conventional techniques, the cellulose istypically recovered and purified before functionalization, whichpresents additional fabrication steps, the use of harsh chemicals,similar challenges in composite uniformity and limitations to nanofiberfunctionalization. Referring now to the drawings, and more particularlyto FIGS. 1 through 10, where similar reference characters denotecorresponding features consistently throughout the figures, there areshown preferred embodiments. In the drawings, the size and relativesizes of components, layers, and regions, etc. may be exaggerated forclarity.

FIG. 1 illustrates an apparatus 5 for producing cellulose-basedstructures according to the workflow of FIG. 2. Bacteria 10 is providedin a container 15. In an example, the container may comprise a culturechamber, such as one made from plastic or glass. According to anexample, the bacteria 10 may comprise Gluconacetobacter. Other types ofbacteria may also be used in accordance with the embodiments herein.Culturing media 20 is provided in the container 15 to sustain thebacteria 10. In some examples, the culturing media 20 may aid in thegrowth of the bacteria 10 in the container 15. Additives 25 are combinedwith the bacteria 10 to produce a cellulose nanofiber product, referredto herein as cellulose nanofiber 30 a. According to an example, thecellulose nanofiber 30 a may be modified during production through anyof diffusion, binding, and surface modification of fibers constitutingthe cellulose nanofiber 30 a. The bacteria 10 fabricates a scalablematrix of nano-cellulose (e.g., cellulose nanofiber 30 a), which is adesirable framework material that presents advantages over its commonlyused alternative-plant-derived cellulose. In an example, the additives25 may comprise organic additives 25 a. In this regard, the organicadditives 25 a may comprise any of compounds, click handle analogs,carbon/oxide particles, biopolymers, and catalytic enzymes. In anexample, the biopolymers may comprise any of proteins, carbohydrates,and nucleic acids. According to another example, the additives 25 maycomprise inorganic additives 25 b. In this regard, the inorganicadditives 25 b may comprise any of metal nanoparticles, metal oxides,and metal salts. For example, nanoparticles can be uniformlyincorporated into the cellulose nanofiber 30 a in a bio-friendly way,without chemical treatments. Instead, the additives 25 (e.g., such asnanoparticles, etc.) are coincubated with the bacteria 10 to distributethem during cellulose generation. Furthermore, the additives 25 (e.g.,such as nanoparticles, etc.) can be readily tethered/functionalized tothe nanofibers using the cellulose-binding fusion proteins. The dottedarrow shown in FIG. 2 represents the direction of cellulose pelliclegrowth at the air/water interface.

The biopolymers such as fusion proteins could be assembled onto thebacterial cellulose nanofiber 30 a and tailored to tether nanoparticlesonto the cellulose nanofiber 30 a. In an example, strontium titanatecould be incorporated into cellulose nanofiber 30 a in a biocompatiblemanner, recovered for sintering, and tested to yield dielectricperformance with desirable high permittivity properties. Alternatively,supplementing bacterial nutrient broth with elements of interest (in theform of salts), which, once carbonized, become doped with the elementsand provide desirable performance as an electrocatalyst for oxygenreduction reactions. The supplements do not deter growth or celluloseaccumulation.

The workflow further includes controlling a concentration of theadditives 25 in the cellulose nanofiber 30 a to create a uniformdistribution of the additives 25 in the cellulose nanofiber 30 a. In anexample, the controlling of the concentration of the additives 25 mayoccur (A) by purifying the cellulose nanofiber 30 a by solvent exchangeto diffuse away extra additives 25. In another example, the controllingof the concentration of the additives 25 may occur (B) by evaporatingthe cellulose nanofiber 30 a to enhance the concentration of theadditives 25 within the cellulose nanofiber 30 a.

Thereafter, the cellulose nanofiber 30 a is processed at a selectedtemperature to create a composite material 35. The processing of thecellulose nanofiber 30 a at the selected temperature may comprisefreeze-drying the cellulose nanofiber 30 a. In an example, thefreeze-drying of the cellulose nanofiber 30 a may create a dehydratedmaterial (e.g., the composite material 35 may be configured as adehydrated material). In another example, the processing of thecellulose nanofiber 30 a at the selected temperature may compriseperforming pyrolysis on the cellulose nanofiber 30 a. The performing ofthe pyrolysis on the cellulose nanofiber 30 a may create a carbonizedmaterial (e.g., the composite material 35 may be configured as acarbonized material). The production of the advanced functionalcomposite material 35 from the cellulose nanofiber 30 a may occur underconditions compatible with the culturing of the Gluconacetobacterbacteria 10.

The workflow creates readily-formed cellulose composite precursors inconjunction with Gluconacetobacter propagation and cellulose productionin the culturing media 20 by utilizing biofabrication principles andstraightforward material recovery steps prior to post-processing.Bacterial cellulose scaffolds are functionalized in situ using additives25 of biocompatible composition and concentration supplemented in thegrowth media 20 upon inoculation. Additives 25 (especially biologicalcomponents) may facilitate binding interactions and hierarchicalassembly of components directly onto the cellulose scaffold (e.g.,cellulose nanofiber 30 a). In this way, the additives 25 readilyincorporate with the cellulose nanofiber 30 a as it is generated.

The cellulose composite material 35 having additives 25 incorporatedtherein may be recovered using solvent exchange or solventconcentration. As indicated in FIG. 2, solvent exchange (A) can be usedwhen scaffold functionalization is desired in order to eliminateunbound, superfluous additives 25, and retaining a composite material 35with immobilized functional components. Conversely, as indicated in FIG.2, solvent concentration (B) allows additives 25 to be enriched withinthe cellulose scaffold at concentrations that do not support bacterialgrowth but are preferred once cellulose has accumulated to a sufficientbiomass. Subsequent post-processing steps are minimal steps (e.g.,freeze-drying for a dehydrated material or pyrolysis for a carbonizedmaterial) to achieve materials suitable for advanced applications.

FIGS. 3A through 3C, with reference to FIGS. 1 and 2, are flow diagramsillustrating a biofabrication method 200. As shown in FIG. 3A, thebiofabrication method 200 comprises providing (205) bacteria 10 such asGluconacetobacter in a container 15; providing (210) culturing media 20in the container 15; and coincubating (215) additives 25 with theGluconacetobacter in the container 15 to produce a moldable cellulosehydrogel 30 b. In an example, the cellulose hydrogel 30 b comprisesentangled bacteria-produced cellulose nanofibers 30 a. The coincubationof the additives 25 with the Gluconacetobacter may occur throughdiffusion, binding, or surface modification of the cellulose nanofibers30 a. Next, the biofabrication method 200 comprises controlling (220) aconcentration of the additives 25 in the cellulose hydrogel 30 b to forma composite precursor material 35; and thermally processing (225) thecomposite precursor material 35. The composite cellulose hydrogel 30 bcan be purified by solvent exchange to diffuse away superfluousadditives 25 or conversely evaporated to enhance the concentration ofthe additives 25 within the cellulose hydrogel 30 b.

In an example, the additives 25 may comprise a chemical compound or saltas a source of nitrogen, iron, phosphorous, sulfur, or other chemicalelement of interest for impregnation or nanoparticle formation.According to an example, the coincubating (215) of the additives 25 withthe Gluconacetobacter in the container 15 may occur at a temperaturerange between approximately 20-30° C. As shown in FIG. 3B, the method200 may comprise forming (230) a metallized aerogel from the thermallyprocessed composite precursor material 35. As shown in FIG. 3C, themethod 200 may comprise forming (235) a doped electrocatalyst from thethermally processed composite precursor material 35. The in situbiofabrication method 200 of the cellulose composite precursors promotesuniform distribution of functional components, takes advantage of thecellulose nanofiber network for autonomous generation of a high surfacearea 3D composite precursor material 35, and as a result, enables asimplified fabrication workflow.

The establishment of binding specificity between constituentsfacilitates hierarchical self-assembly (e.g., affinity interactions,nucleic acid hybridization, biorthogonal click chemistry, etc.), withsimple elimination of superfluous unassembled constituents from thecellulose hydrogel by solvent exchange. Alternatively, by evaporatingthe culture media 20, the additive 25 reaches a desired concentrationthat is uniformly distributed within the cellulose hydrogel 30 b. Thus,both solvent-exchanged and solvent-concentrated cellulose hydrogelspresents a composite precursor material 35 that can be obtained via thetwo-step process (e.g., biofabrication and recovery). The bacterialcellulose hydrogel 30 b can be readily customized into molded shapes bythe corresponding shape and configuration of the culturing container 15and uniformly biofunctionalized with fusion proteins, uniquely providingnanoscale visual resolution of the “living” material and nanofiberfunctionality.

Since the steps of the biofabrication method 200 occur autonomously andin an ambient environment (e.g., cellulose accumulation, bindinginteractions for self-assembly, passive diffusion and incorporation inaqueous solution and room temperature), the method 200 simplifies thefabrication process for several materials (e.g., with dielectrics andelectrocatalysts, etc.). Conventional biofabrication workflows mayrequire user-based fabrication of a polymeric aerogel framework, surfacechemistry and chemical grafting protocols, and organic/aqueous solventexchanges with challenges in scalability, shaping, and compositeuniformity. Additionally, in conventional techniques that use bacterialcellulose scaffolds, the cellulose is typically recovered and purifiedbefore functionalization, which presents additional fabrication steps,the use of harsh chemicals, similar challenges in composite uniformity,and limitations to nanofiber functionalization, all of which suchlimitations are overcome using the biofabrication method 200 provided bythe embodiments herein.

FIG. 4, with reference to FIGS. 1 through 3C, is a flow diagram of abiofabrication method 300 for producing cellulose-based materials. Themethod 300 comprises providing (305) active cellulose-producing bacteria10 and culturing media 20 in a container 15; combining (310) organicadditives 25 a or inorganic additives 25 b with the activecellulose-producing bacteria 10 in the container 15 to produce acellulose hydrogel matrix 30 c composed of entangled bacteria-producedcellulose nanofibers 30 a; controlling (315) a concentration of theadditives 25 in the cellulose hydrogel matrix 30 c; and exposing (320)the cellulose hydrogel matrix 30 c in selected thermal environment tocreate a biofabricated functional material (e.g., composite material35). To customize the cellulose functionality for various applications,functional moieties (e.g., organic additives 25 a or inorganic additives25 b such as nanoparticles, etc.) are either bound directly to thematrix fibers (e.g., using fusion proteins) or passively incorporated asthe bacteria 10 produce the cellulose hydrogel matrix 30 c. Uponrecovery of the cellulose hydrogel matrix 30 c, the solution components(e.g., the additives 25 and entangled bacteria-produced cellulosenanofibers 30 a) can be further concentrated into the cellulose hydrogelmatrix 30 c by evaporating the solution. Thus, preparation of theprecursor composite material 35 includes a set-up of the bacteria 10 inthe culturing media 20 containing desired components for the compositematerial 35, and specialized recovery of the composite material 35 uponits in situ formation. The final composite material 35 utilizes onlyminimal post-processing steps for typical downstream applications suchas metallized aerogels, doped electrocatalysts, etc. (e.g.,freeze-drying for dehydration or pyrolysis to carbonize the compositematerial 35).

The biofabrication method 300 and recovery steps produce advancedfunctional materials (e.g., composite material 35) from bacterialcellulose (e.g., cellulose hydrogel matrix 30 c composed of entangledbacteria-produced cellulose nanofibers 30 a). The biofabrication can bedefined as a process of using biomaterial building blocks (e.g., cellsand biopolymers like proteins, nucleic acids, and polysaccharides) andbenign assembly methodologies (e.g., binding interactions, enzymaticgrafting) that occur in ambient solvents and temperatures to retainbiological functionality. In this case, cellulose composite precursorscan be readily formed in conjunction with Gluconacetobacter propagationand cellulose production in preferred growth culturing media 20. This insitu fabrication of cellulose composite precursors promotes uniformdistribution of functional components, takes advantage of the cellulosenanofiber network for autonomous generation of a high surface area 3Dmaterial, and as a result, enables a simplified fabrication workflow.Protein and peptide binding interactions with both cellulose nanofibers(e.g., fusion proteins containing a cellulose binding module (CBM) suchas one derived from Clostridium thermocellum) and functional components(e.g., inorganic-binding peptides, peptides that promote mineralization)can be used to functionalize cellulose nanoscaffolds with inorganicparticles (e.g., ferromagnetic, dielectric particles). Thesupplementation of growth culturing media 20 with added elements,combined with post-growth media concentration as the recovery step,establishes a novel strategy to generate superior precursors for dopedcarbonized aerogels while supporting bacterial culture growth. Theunique biofabrication strategies yield material precursors that arechallenging to fabricate by other methods, and when post-processed,demonstrate functional properties (e.g., high dielectric permittivity,electrocatalytic oxygen reduction efficiency) that are superior topreviously-reported metrics for similar, conventional materials.

Gluconacetobacter shaking cultures may be used with added nanoparticles(such as, for example, strontium titanate (ST) or iron oxide (Fe₃O₄))for direct incorporation into the cellulose hydrogel 30 b. FIG. 5, withreference to FIGS. 1 through 4, demonstrates the enhanced bindingaffinity of ST and Fe₃O₄ particles to cellulose hydrogels 30 b byincluding His10-sfGFP-CBM fusion protein with affinity to both thenanoparticles (via His10) and cellulose nanofibers 30 a (via CBM),imaged by brightfield microscopy with magnified inset images.

The resulting biofabricated cellulose-nanoparticle composite material 35is scalable to achieve functional materials having desirable properties.In an example, biofabricated cellulose-ST is formed into a capacitormaterial with a measured dielectric constant that is superior to valuesof conventional bulk materials. FIGS. 6A and 6B, with reference to FIGS.1 through 5, depict scanning electron micrographs of (FIG. 6A) ST powderand (FIG. 6B) a sintered ST-cellulose composite sample (prepared at1300° C. under inert atmosphere for 1+h). FIG. 6C, with reference toFIGS. 1 through 6B, illustrates stoichiometric ratios of the atomicpercentage of strontium (Sr) to titanium (Ti) (analyzed by energydispersive x-ray spectroscopy) for sintered samples prepared either STentrapped in an inert hydrogel (using 5, 50, or 500 mg/ML ST) or STentrapped in cellulose via bacterial culturing (using 10 mg/ML ST). FIG.6D, with reference to FIGS. 1 through 6C, illustrates an example of abulk dielectric material made by filling ST-cellulose withpolydimethylsiloxane (PDMS) using the techniques provided by theembodiments herein.

The workflow 100 and biofabrication methods 200, 300 simplify the dopingconditions by coincubation of additives 25 (e.g. such as urea and ironchloride for N and Fe doping) with the bacteria 10 such asGluconacetobacter, without the need for material purification andimpregnation with chemicals. This provides a bio-friendly approach, andmaintains efficient cellulose production as indicated by theexperimental results shown in FIGS. 7A through 7C, with reference toFIGS. 1 through 6D. FIG. 7A depicts timecourse monitoring ofGluconacetobacter productivity through measurements of glucose depletionrate and cellulose biomass accumulation rate. FIG. 7B illustratesmeasurements of a cellulose biomass produced by cultures containingvarious elemental additives 25 (e.g., urea, iron chloride (Fe), sodiumphosphate (P), and sodium sulfate (S)). FIG. 7C illustrates a plot ofcellulose mass vs. residual glucose in a nutrient media 20 for cultureswith elemental additives 25.

Using the techniques provided by the embodiments herein, both the bulkstructure (molded 3D shapes) and porous nanostructure may be retained incarbonized bacterial cellulose as demonstrated in FIGS. 8A through 8E,with reference to FIGS. 1 through 7C. FIG. 8A illustrates measurementsof a cellulose mass after freeze-drying to determine the water contentof cellulose hydrogel and of a carbonized product after pyrolysis. FIG.8B is an image of representative cellulose hydrogel shapes and FIG. 8Cis a scanning electron micrograph of cellulose after freeze-drying. FIG.8D is an image of representative cellulose shapes after pyrolysis andFIG. 8E is a scanning electron micrograph of the resulting carbonizednanonetwork.

Through material characterization, experimental results are demonstratedin effectively and uniformly doped carbon networks, as shown in FIGS. 9Athrough 9C, with reference to FIGS. 1 through 8E. FIG. 9A illustrateselemental composition of cellulose derived mesoporous carbons, asdetermined by energy-dispersive X-ray spectroscopy (EDS). Catalyticactive elements are plotted, with the residual mostly carbon with someoxygen and salt. FIG. 9B is a SEM image of Fe—S, P, N carbons. Thearrows draw attention to several nanoparticles formed during processing.FIG. 9C illustrates XPS data for urea-doped and undoped cellulosederived carbons. Almost no residual nitrogen is observed in the undopedsample while the urea doped sample has a peak at 398 eV, indicative ofcatalytically active pyridinic nitrogen.

Electrocatalyst materials traditionally require precious metals (e.g.,platinum, etc.) for high density energy technologies like fuel cells andbatteries. Biofabricated doped carbonized bacterial cellulose provides amuch cheaper metal-free alternative electrocatalyst material withelectrochemical performance that matches a platinum standard. FIG. 10,with reference to FIGS. 1 through 9C, illustrates cyclic voltammagramsobtained on cellulose-derived carbons that where various chemicalsduring growth to impart particular elements to the resulting material.The undoped sample is prepared using the Gluconacetobacter in aculturing growth media 20, while the other three traces have been dopedwith: nitrogen (urea), sulfur, phosphorous and nitrogen (S, P, N), oriron along with the other elements (Fe—S, P, N).

The Gluconacetobacter bacteria 10 may be used to produce a bulkcomposite material 35 with the desired porosity and nanofiber aspectratios. The in situ functionalization through the biofabricationworkflow 100 and methods 200, 300, which occur in ambient conditionswith minimal user intervention, eliminate the purification andcrosslinking/grafting steps inherent in the conventional solutions. Byin situ biofabrication, functionalization occurs as the cellulosenanofiber 30 a is directly generated by the bacteria 10, resulting in auniform composite material 35. Moreover, the use of biomolecules forfunctionalization supply many binding interactions with high specificitythat enables fine-tuned control, patterning, and hierarchical assembly.

The techniques provided by the embodiments herein are highly versatilefor functionalization of porous nanofiber scaffolds with biologicalmoieties, nanoparticles, and elements. The combination of cellulose withcustomized functional groups could benefit a variety of applicationsincluding renewable energy conversion and high-density storagetechnology (e.g., fuel cells, batteries, supercapacitors). Furthermore,the embodiments herein may be used for next-generation conformalmaterials used for interacting with electromagnetic wavelengths(absorption, transmission, etc.) for vehicles, robots, etc.Additionally, the embodiments herein may be used for filters and/orcatalysts for protection against and remediation of contaminants orbio/chemical agent neutralization. Moreover, the embodiments herein maybe used for biomedical dressings for tissue engineering and woundtreatment, according to various examples.

The embodiments herein enable fabrication of new iterations ofcommercial and military-relevant materials and for point-of-needpreparation. Oxygen reduction reaction (ORR) catalyst and highemissivity (c) materials may be produced using the techniques providedby the embodiments herein and provide applications of energystorage/conversion, and materials for electronic warfare devices.Specifically, the ORR catalyst has direct application for anion exchangemembrane (AEM) fuel cells and batteries (e.g., Zn-Air) and demonstratesthat general utility towards preparing electrocatalysts, which can findapplications beyond energy storage, for example, chemical agentamelioration. The embodiments herein provide a “green” workflow thateliminates the use of harsh chemicals, minimizes processing steps, anddoes not need to use nanoparticles or other additives in excess sincethey are directly incorporated in situ.

The templating of SrTiO₃ nanostructures allows lightweight, highdielectric constant materials for next generation commercial andmilitary equipment and vehicles. Furthermore, the embodiments herein areapplicable for many types of nanoparticles, with related compounds (e.g.barium titanate (BT) and barium strontium titanate (BST), etc.) beingspecifically useful for capacitors for high-power applications. Inaddition to the applications enabled by the embodiments herein,bacterial cellulose has a range of attractive properties, including lowweight, high mechanical toughness and good thermal insulation that makeit an attractive platform for commercial and military manufacturing inresource limited environments. For example, the development of highdielectric materials is also of considerable commercial interest,especially when integrated with conventional and 3D printing-basedprocessing.

The embodiments herein provide a technique for the preparation ofcomposite materials 35 by using a bacterial cellulose source (e.g.,cellulose nanofiber 30 a, cellulose hydrogel 30 b, or cellulose hydrogelmatrix 30 c) that provides an aerogel/high surface area framework, whichis a deviation from conventional solutions where cellulose fibers fromplants are crosslinked to generate such structures. Moreover, theembodiments herein have utility for “low profile” conformal materialsfor energy storage/conversion and electromagnetic signal maintenance.The autonomous, in situ biofabrication approach enables such materialsto be assembled remotely, such as from a minimal bacterial inoculum“seed” that can be scaled up at the point of need. This approach willalso continue to leverage and intersect with the emerging field ofgenetic engineering/synthetic biology to facilitate complexity in thebiofabrication steps performed by a living cellulose-producing cultureof bacteria, such as spatiotemporal patterning.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the embodiments herein that others may, byapplying current knowledge, readily modify and/or adapt for variousapplications such specific embodiments without departing from thegeneric concept, and, therefore, such adaptations and modificationsshould and are intended to be comprehended within the meaning and rangeof equivalents of the disclosed embodiments. It is to be understood thatthe phraseology or terminology employed herein is for the purpose ofdescription and not of limitation. Therefore, while the embodimentsherein have been described in terms of preferred embodiments, thoseskilled in the art will recognize that the embodiments herein may bepracticed with modification within the spirit and scope of the appendedclaims.

What is claimed is:
 1. A method of producing cellulose-based structures,the method comprising: providing bacteria in a container; providingmedia in the container to sustain the bacteria; combining additives withthe bacteria to produce a cellulose nanofiber; controlling aconcentration of the additives in the cellulose nanofiber to create auniform distribution of the additives in the cellulose nanofiber; andprocessing the cellulose nanofiber at a selected temperature to create acomposite material.
 2. The method of claim 1, wherein the bacteriacomprises Gluconacetobacter.
 3. The method of claim 1, wherein theadditives comprise organic additives.
 4. The method of claim 3, whereinthe organic additives comprise any of compounds, click handle analogs,carbon/oxide particles, biopolymers, and catalytic enzymes.
 5. Themethod of claim 4, wherein the biopolymers comprise any of proteins,carbohydrates, and nucleic acids.
 6. The method of claim 1, wherein theadditives comprise inorganic additives.
 7. The method of claim 6,wherein the inorganic additives comprise any of metal nanoparticles,metal oxides, and metal salts.
 8. The method of claim 1, wherein thecellulose nanofiber is modified during production through any ofdiffusion, binding, and surface modification of fibers constituting thecellulose nanofiber.
 9. The method of claim 1, wherein the controllingof the concentration of the additives occurs by purifying the cellulosenanofiber by solvent exchange to diffuse away extra additives.
 10. Themethod of claim 1, wherein the controlling of the concentration of theadditives occurs by evaporating the cellulose nanofiber to enhance theconcentration of the additives within the cellulose nanofiber.
 11. Themethod of claim 1, wherein the processing of the cellulose nanofiber atthe selected temperature comprises freeze-drying the cellulosenanofiber.
 12. The method of claim 11, wherein the freeze-drying of thecellulose nanofiber creates a dehydrated material.
 13. The method ofclaim 1, wherein the processing of the cellulose nanofiber at theselected temperature comprises performing pyrolysis on the cellulosenanofiber.
 14. The method of claim 13, wherein the performing of thepyrolysis on the cellulose nanofiber creates a carbonized material. 15.A biofabrication method comprising: providing Gluconacetobacter in acontainer; providing culturing media in the container; coincubatingadditives with the Gluconacetobacter in the container to produce acellulose hydrogel; controlling a concentration of the additives in thecellulose hydrogel to form a composite precursor material; and thermallyprocessing the composite precursor material.
 16. The method of claim 15,wherein the additives comprise a chemical compound or salt as a sourceof nitrogen, iron, phosphorous, sulfur, or other chemical element ofinterest for impregnation or nanoparticle formation.
 17. The method ofclaim 15, comprising forming a metallized aerogel from the thermallyprocessed composite precursor material.
 18. The method of claim 15,comprising forming a doped electrocatalyst from the thermally processedcomposite precursor material.
 19. The method of claim 15, wherein thecoincubating of the additives with the Gluconacetobacter in thecontainer occurs at a temperature range between approximately 20-30° C.20. A biofabrication method for producing cellulose-based materials, themethod comprising: providing active cellulose-producing bacteria andculturing media in a container; combining organic additives or inorganicadditives with the active cellulose-producing bacteria in the containerto produce a cellulose hydrogel matrix composed of entangledbacteria-produced cellulose nanofibers; controlling a concentration ofthe additives in the cellulose hydrogel matrix; and exposing thecellulose hydrogel matrix in selected thermal environment to create abiofabricated functional material.